This is a continuation of co-pending international application No. PCT/FR99/02462 filed on Oct. 12, 1999, which designated the United States of America.
The present invention relates to integrated periodontal implants comprising cementum on the surface of the root and a ligament to connect the cementum to the bone alveolus, as in a natural tooth.
The invention pertains to a method for preparing the implant by bringing the implant into contact with undifferentiated mesenchymal stem cells under culture conditions to allow adhesion of cementoblasts and of alveolodental ligament to the root portion of the implant and implanting the implant carrying differentiated tissue cells.
The invention also relates to a cell culturing apparatus for preparing a dental implant. Finally, it relates to a method for replacing lost or compromised teeth with implants to which biological tissues and cells have been affixed using a suitable cell culture technique, to then obtain mouth cicatrisation by cementum and ligaments between the bone alveolus and implanted roots.
Dental transplants and implants have been carried out for several decades and a variety of techniques have been described.
Implanting techniques in current use include the following:
a) prostheses on osteo-integrated implants, artificial titanium roots which are stabilized by bony ankylosis. The force of mastication is transmitted to the bone with no dampening because of the absence of alveolodental ligament which is an important factor in protecting teeth against shock, overload and the risk of fracture;
b) transplants or other dental grafts which do not have the disadvantages of the preceding approaches but require extraction of a donor tooth which is available and generally non-functional.
Before embarking on the description of the present invention, a reminder of the physiological conditions regarding the connections of the natural tooth is necessary. The tooth is anchored in a cavity termed the alveolus, in the alveolar bone. The root and bone are anchored together by a ligament principally constituted by bundles of collagen fibers with one end anchored in the bone and the other end in the cementum, a mineralised layer resulting from differentiation of undifferentiated mesenchymal cells to cementoblasts which produce the organic and mineral matrices constituting the cementum. Collagen fibers are perpendicularly inserted in this cementum, and are included parallel to the cementum surface, forming a network.
In order to be functional and accepted, ideally, implanting an artificial tooth must anatomically and histologically reproduce the support structures of natural teeth, i.e., the cementum, the alveolodental ligament, and the alveolar bone, with all of their components: differentiated or undifferentiated cells, collagenic fibers and other fibers (elastic, oxytalan, elauin), the basic substance, mineralised tissues, vascularisation and innervation.
The patent application EP-A-734712 (Kanebo Ltd) describes a method for carrying out an implant by applying a layer of cementum particles to the surface. However, such implant does not present collagen fibers perpendicularly inserted in the surface of the neoformed cementum, necessary to the attachment of said implant with the alveolar cavity.
In order to stimulate the reformation of alveolodental ligaments on the curetted dental roots, Hanes et al. (xe2x80x9cCell and Fiber attachment to demineralised cementum from normal root surfacesxe2x80x9d Vol. 60, no. 4, pages 188-198), have studied the effect of citric acid on its surface; the cementum or dentin fibers removed by demineralisation can bind by xe2x80x9csplicesxe2x80x9d to the collagen fibers in the surrounding tissues. But there is no xe2x80x9cneo-cementxe2x80x9d and the fibers stand much less dense than a normal desmodont The fibroblasts, ligament generators, cementoblasts and osteoblasts bordering the alveolar bone result from differentiation of undifferentiated mesenchymal stem cells which are normally sited in the connective tissues surrounding the blood vessels. They can be found on the surface of the roots of extracted teeth and/or in the alveolus of extracted teeth or in the ligaments of a tooth or in other tissues, which may or may not be buccal connective tissue.
The invention results from a demonstration that under certain biological and mechanical stimulation conditions, the natural physiological environment of the root described above can be reconstituted from a culture of undifferentiated mesenchymal stem cells.
The proposed invention can thus enable teeth lost in the majority of clinical situations to be replaced with permanent artificial teeth connected to the jaws by the same tissue elements as natural teeth, i.e., a cementum, an alveolodental ligament and an alveolar bone and enabling normal attachment of the gingivae to the neck of the implant. These four elements are constituents of the periodontal tissue which can thus fulfil its normal physiological role, namely dampening the stresses of mastication, preventing overloads, and adapting the position and mobility of the tooth to the average load it receives.
The present invention provides a method of manufacturing a dental implant comprising:
preparing an implant composed of a root portion and a crown portion and constituted by a biocompatible material in a shape which is adapted to an extracted tooth;
immersing the root portion of said implant in a culture of undifferentiated mesenchymal stem cells in a culture medium the composition of which allows differentiation into cementoblasts and fibroblasts, over a period which is sufficient for said differentiation and for adhesion of cementoblasts to the root portion and the formation of a first layer of cementum and an alveolodental ligament primordium attached to said cementum;
recovering the implant carrying differentiated tissues affixed to its root portion.
The choice of implant is governed by different criteria. The first criterion is its morphology. This depends on the volume of dentin (ivory) in the tooth to be replaced, i.e., the total volume less the enamel and cementum. The desired shape is obtained, for example, by comparison with the extracted tooth; it is then customised to the exact shape of the tooth to be replaced. The desired shape can also be obtained from radiological or tomodensitometric data or the like. The implant is then shaped from a block constituted by a selected material using an N/C machine or any other means. A few basic shapes may also be satisfactory in a majority of cases; they will have a variety of lengths, diameters or tapers, with round or oval cross sections.
The choice of implant material in the method of the invention is guided firstly by the biological and immunological acceptability of said material in the mouth, and secondly by its performance as a support for cementoblast adhesion. The material of the implants must be biocompatible and must be sufficiently strong mechanically to avoid the risk of fracture; the surface must also be roughened to facilitate cell adhesion, and finally its color must be close to that of the natural tooth. Known materials can be envisaged for the material of the implant, such as titanium, alloys or ceramics, for example zirconia. Natural devitalised teeth obtained from any source may also be used. Such materials have been described in Periodontology 2000 (1998) 17: 7-21.
In addition to the root portion which is brought into contact with the undifferentiated mesenchymal culture, the dental implant used in the method of the invention comprises a crown portion which can be capped by a crown of resin, a composite, or a metal or ceramic alloy. Grooves are provided in the crown portion of the implant to stabilise the sutures which hold the implant in its alveolus on placing it in the mouth. With the implant viewed occlusally a groove can, for example, occupy one diameter and two others can cross this diameter at a right angle at a point equidistant from the center and from the circumference. However, these grooves can be disposed differently without disturbing the ergonomics of the system, as will be shown in Example 3 below.
Finally, the implant used in the method of the invention can comprise a stent which is coaxial with the tooth and fixed on its crown portion to facilitate manipulation and subsequent re-implanting in the mouth.
One of the essential characteristics of the method of the invention is the formation, by in vitro cell culture in the presence of the implant the shape and material of which has been selected using the criteria described above, of a layer of cells and tissues adhering to the root portion of the implant. These cells and tissues exist naturally between the root of the tooth and the alveolar bone; thus the physiology of re-implanting in the mouth is very similar to natural conditions.
It is well known that fibroblasts are the dominant cell type in all connective tissues in the human body and in particular are the essential cells of the tissues of the periodontal ligament. The differentiated cells of the periodontal tissue, namely cementoblasts, fibroblasts and osteoblasts, originate from differentiation of undifferentiated cells which are the mesenchymal stem cells. These cells are normally found in the connective tissues surrounding the blood vessels. They can be removed from the surface of the roots of extracted teeth if they have not been contaminated and/or from the alveolus of extracted teeth, or from the ligament of a tooth by removal using a trocar needle, or from explants of buccal connective tissue.
These stem cells exist in other connective tissues of the body, and can thus be removed from other, non-buccal, sites.
The removed undifferentiated cells are cultured under conventional conditions, for example in a petri dish, in Falcon type culture dishes, or in rolling bottles.
The removed undifferentiated mesenchymal cells are cultured in a medium the composition of which enables growth and differentiation into fibroblasts and cementoblasts. Such media are conventional media for culturing fibroblastic animal cells such as those described, for example, in the review article by S. Pitaru et al., in J. Periodont. Res. (1994), 29: 81-94. By way of example, DMEM medium (Dulbecco""s Modification of Eagle""s MEM) (Dulbecco and Freeman, 1959: Morton, 1970) supplemented with foetal calf serum can be used. This medium is supplemented with antibiotics and antifungal agents. The culturing cells are biologically stimulated by adding to the medium the molecules or compositions required for development and differentiation of the cementoblasts and fibroblasts. They may be growth factors. Examples which can be cited are: PDGF, IGF, proteins from the embryonic enamel organ, bFGF, and other molecules having an anabolising effect on periodontal tissue, for example nifedipine, vitamin C or avocado, maize and/or soya nonsaponifiable matter. This list is not limiting.
The patent application WO 97/45533 (Rutherford) discloses methods for regenerating different tissues and in particular, oral and dental tissues using ex vivo culture of cells. More specifically, it relates to a culture of cementum cells or cells derived from the alveolodental ligament. It also relates to the use of structural matrix to enable tissue differentiation.
When the cell density reaches 105 cells/ml, the cells are transferred to a culturing apparatus for immersion of the root portion of the implant to be treated. The appropriate culturing apparatus also forms part of the invention and is described below.
The implant is then positioned in the culturing apparatus until a layer of cementoblasts adhering to its root portion is obtained which produces a first layer of cementum with connective fibers inserted in this cementum, and a second layer containing fibroblasts and collagen in the course of being formed.
After immersion in the culture medium for 15 to 30 days in the apparatus, the implant is then recovered and xe2x80x9cimplantedxe2x80x9d in the mouth in the alveolus under the conditions described in the protocol of Example x below.
In the method of the invention, biological stimulation of cell differentiation of the undifferentiated mesenchymal cells can be completed by xe2x80x9cphysiologicalxe2x80x9d mechanical stimulation. This is effected by applying a periodical force to the implant when it is immersed in the culturing cell. This can also be achieved by a periodical stirring applied to the culture apparatus, the implant being then fixed by any appropriate means. One should apply a moderate motion between the implant and the artificial alveolus included in the apparatus containing the culture medium in which the implant is immersed. Stirring can be an alternating motion with a period in the range 1 to 60 seconds without restricting this method to that range, and with an amplitude in the range 0.005 to 2 mm; the displacement can be horizontal, i.e., orthogonal to the axis of the implant, vertical, i.e., longitudinal to the axis of the implant, or a combination of the two. It can also be a rotary motion.
The motion applied to the implant in its culture medium has a double function, conferred by the relative motion between the implant and the artificial alveolus formed by the porous membrane. The first function is to create a functional stimulation of the cells under culture, which increases their proliferation, differentiation, synthesising activities (cementum and collagen) and physiological orientation of the structures being formed. The second function of this agitation is to agitate the cell culture, as it is recommended when culturing any eukaryotic cells; this aerates the cells better and also circulates the medium and nutrients in the cell environment, encouraging their development.
This agitation can be effected using any system which can adjust the period and amplitude within the limits defined above. It may be a mechanical, electrical, hydraulic or pneumatic system, this list not being limiting. Different embodiments can be envisaged in this regard: the motion can be applied to the implant alone, via a stent which is fixed to crown portion of the implant; it can also be applied to the system assembly containing the cell culture in which the root portion of the implant is immersed. Finally, it can be applied to cell culture, the implant proper being fixed to a support by a stent which is integral with the crown portion. What is desired in this mechanical agitation is a relative motion of the root portion of the implant and the culture medium in which it is immersed, with the period and amplitude conditions described above which can satisfy the two functions, cell stimulation and cell agitation.
The present invention also relates to a cell culturing apparatus for preparing a dental implant.
More particularly, when referring to FIG. 1, the present invention provides a cell culturing apparatus for preparing a dental implant (30) constituted by a root portion (31) and a crown portion on which a stent (32) is fixed to enable it to be manipulated and subsequently placed in the mouth, said apparatus comprising:
a culture dish (10) the shape of which comprises a longitudinal axis;
said culture dish being closed by a cover (11) and comprising a porous wall (20) delimiting a first space (21) containing the cell culture medium, and a second space (22) containing the cells being cultured, and in which the root portion of the implant is immersed, said wall having a configuration such that a gap of 0.1 to 5 mm, ideally 1 mm, is left between the root portion of the implant and the wall, creating an artificial alveolus.
Said apparatus comprises a means for agitating the implant in said artificial alveolus using an alternating motion with a period in the range 5 to 60 seconds and with an amplitude in the range 0.005 to 2 mm, or in a rotary motion with a period in the range 1 to 60 seconds. This agitation means can be mechanical, hydraulic or magnetic to confer an alternating or rotary motion on the stent. Alternatively, the apparatus may comprise a means for agitating of the culture dish and a means for maintaining the dental implant.
The present invention also relates to a dental implant which can be obtained by a method as described above and which can reconstitute, on the root surface of the implant, the different constituent tissues of the periodontal tissue, namely the cementum and the ligaments connecting the cementum to the alveolar bone. This implant or artificial tooth is constituted by an inert biocompatible material the shape of which is adapted to that of the extracted tooth. The dental implant of the invention will thus comprise a crown portion and a root portion which, before implanting in the mouth, will be coated with differentiated cells which can then allow more complete formation of the cementum and ligament in situ. A dental implant of the invention can ensure complete reconstitution of a normal periodontal tissue system supporting the implant in two to three months.
Finally, the invention provides a method for replacing lost or compromised teeth by artificial implants on which biological tissues are affixed using a suitable cell culture technique, to then obtain mouth cicatrisation by cementum and ligament between the bone aveolus and implanted roots.